Pro-inflammatory properties of M1 phenotypes of human macrophages: prolongation of myeloperoxidase-mediated oxidative stress.

Abstract

Macrophages and neutrophils are the main immune cells of the acute stage of inflammation. Upon their activation, membrane-bound NADPH oxidase produces superoxide anion radical, which is converted to H₂O₂ by superoxide dismutase (SOD). In this study, we compared the production of hydrogen peroxide by two phenotypes of pro-inflammatory human M1 macrophages and neutrophils activated with phorbol-12-myristate 13-acetate. Macrophages were obtained from blood monocytes (monocyte-derived macrophages (MDM)) differentiated into MDM using GM- or M-CSF growth factors and polarized into the M1 state, receiving GM_M1, M_M1, respectively. The total level of H₂O₂ production measured in the presence of horseradish peroxidase differed significantly between two types of macrophages. Only GM_M1 macrophages had a level of H₂O₂ production comparable to neutrophils. GM_M1 appear at the site of inflammation after neutrophils, they continue the work of neutrophils in creating a pro-inflammatory environment: they produce several times more H₂O₂ and pro-inflammatory cytokines than M_M1, which arrive at inflammatory site later. Upon activation, MDM_M1 formed big blot-like and smaller dense spheroid-like aggregates. Activated neutrophils secrete the enzyme myeloperoxidase (MPO), which synthesizes the very potent oxidant hypochlorous acid (HOCl) only in the presence of H₂O₂. Neutrophils are short lived cells, MPO can use H₂O₂ produced by activated cultured MDM to synthesize HOCl at physiologically relevant concentrations to prolong oxidative stress.